RESUMEN
Inhibitors of VEGF signaling can block angiogenesis and reduce tumor vascularity, but little is known about the reversibility of these changes after treatment ends. In the present study, regrowth of blood vessels in spontaneous RIP-Tag2 tumors and implanted Lewis lung carcinomas in mice was assessed after inhibition of VEGF receptor signaling by AG-013736 or AG-028262 for 7 days. Both agents caused loss of 50%-60% of tumor vasculature. Empty sleeves of basement membrane were left behind. Pericytes also survived but had less alpha-SMA immunoreactivity. One day after drug withdrawal, endothelial sprouts grew into empty sleeves of basement membrane. Vessel patency and connection to the bloodstream followed close behind. By 7 days, tumors were fully revascularized, and the pericyte phenotype returned to baseline. Importantly, the regrown vasculature regressed as much during a second treatment as it did in the first. Inhibition of MMPs or targeting of type IV collagen cryptic sites by antibody HUIV26 did not eliminate the sleeves or slow revascularization. These results suggest that empty sleeves of basement membrane and accompanying pericytes provide a scaffold for rapid revascularization of tumors after removal of anti-VEGF therapy and highlight their importance as potential targets in cancer therapy.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Insulinoma/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Actinas/metabolismo , Inhibidores de la Angiogénesis/farmacología , Animales , Anticuerpos Monoclonales/farmacología , Axitinib , Membrana Basal/efectos de los fármacos , Membrana Basal/metabolismo , Membrana Basal/patología , Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patología , Carcinoma Pulmonar de Lewis/irrigación sanguínea , Carcinoma Pulmonar de Lewis/patología , Colágeno Tipo IV/inmunología , Colágeno Tipo IV/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Imidazoles/farmacología , Imidazoles/uso terapéutico , Indazoles/farmacología , Indazoles/uso terapéutico , Insulinoma/irrigación sanguínea , Insulinoma/patología , Inhibidores de la Metaloproteinasa de la Matriz , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neoplasias/irrigación sanguínea , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Compuestos Orgánicos/farmacología , Pericitos/efectos de los fármacos , Pericitos/metabolismo , Pericitos/patología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Resultado del Tratamiento , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The vasculature of the central nervous system (CNS) is highly specialized with a blood-brain-barrier, reciprocal neuroepithelial-endothelial cell interactions and extensive pericyte coverage. Developmentally, numerous important signaling pathways participate in CNS angiogenesis to orchestrate the precise timing and spatial arrangement of the complex CNS vascular network. From a therapeutic standpoint, the CNS vasculature has attracted increased attention since many human ailments, such as stroke, retinopathy, cancer and autoimmune disease are intimately associated with the biology of CNS blood vessels. This review focuses on growth factor pathways that have been shown to be important in developmental CNS vascularization through studies of mouse genetic models and human diseases.
Asunto(s)
Sistema Nervioso Central/irrigación sanguínea , Sistema Nervioso Central/patología , Regulación del Desarrollo de la Expresión Génica , Neovascularización Fisiológica , Animales , Vasos Sanguíneos/metabolismo , Barrera Hematoencefálica , Movimiento Celular , Humanos , Integrinas/metabolismo , Ligandos , Ratones , Modelos Genéticos , Vasos Retinianos/metabolismo , Transducción de Señal , Proteínas Wnt/metabolismoRESUMEN
Malignant pleural mesothelioma is an aggressive tumor of the pleura with an overall poor prognosis. Even with surgical resection, for which only a subset of patients are eligible, long term disease free survival is rare. Standard first-line systemic treatment consists of a platinum analog, an anti-metabolite, and sometimes anti-angiogenic therapy, but there is currently no well-established standard therapy for refractory or relapsed disease. This review focuses on efforts to develop improved systemic therapy for the treatment of malignant pleural mesothelioma (MPM) including cytotoxic systemic therapy, a variety of tyrosine kinase inhibitors and their downstream effector pathways, pharmacologic targeting of the epigenome, novel approaches to target proteins expressed on mesothelioma cells (such as mesothelin), arginine depletion therapy, and the emerging role of immunotherapy. Overall, these studies demonstrate the challenges of improving systemic therapy for MPM and highlight the need to develop therapeutic strategies to control this disease.
RESUMEN
Although blood-brain barrier (BBB) compromise is central to the etiology of diverse central nervous system (CNS) disorders, endothelial receptor proteins that control BBB function are poorly defined. The endothelial G-protein-coupled receptor (GPCR) Gpr124 has been reported to be required for normal forebrain angiogenesis and BBB function in mouse embryos, but the role of this receptor in adult animals is unknown. Here Gpr124 conditional knockout (CKO) in the endothelia of adult mice did not affect homeostatic BBB integrity, but resulted in BBB disruption and microvascular hemorrhage in mouse models of both ischemic stroke and glioblastoma, accompanied by reduced cerebrovascular canonical Wnt-ß-catenin signaling. Constitutive activation of Wnt-ß-catenin signaling fully corrected the BBB disruption and hemorrhage defects of Gpr124-CKO mice, with rescue of the endothelial gene tight junction, pericyte coverage and extracellular-matrix deficits. We thus identify Gpr124 as an endothelial GPCR specifically required for endothelial Wnt signaling and BBB integrity under pathological conditions in adult mice. This finding implicates Gpr124 as a potential therapeutic target for human CNS disorders characterized by BBB disruption.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Células Endoteliales/metabolismo , Glioblastoma/genética , Infarto de la Arteria Cerebral Media/genética , Hemorragias Intracraneales/genética , Receptores Acoplados a Proteínas G/genética , Uniones Estrechas/metabolismo , Animales , Barrera Hematoencefálica/ultraestructura , Modelos Animales de Enfermedad , Células Endoteliales/ultraestructura , Matriz Extracelular/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Glioblastoma/metabolismo , Infarto de la Arteria Cerebral Media/metabolismo , Hemorragias Intracraneales/metabolismo , Ratones , Ratones Noqueados , Microscopía Electrónica , Microvasos , Pericitos/ultraestructura , Reacción en Cadena en Tiempo Real de la Polimerasa , Uniones Estrechas/ultraestructura , Vía de Señalización WntRESUMEN
Abnormal endothelial proliferation and angiogenesis may contribute to brain arteriovenous malformation (BAVM) formation. G protein-coupled receptor 124 (GPR124) mediates embryonic CNS angiogenesis; thus we investigated the association of single nucleotide polymorphisms (SNPs) and haplotypes in GPR124 with risk of BAVM. Ten tagging SNPs spanning 39 kb of GPR124 were genotyped in 195 Caucasian BAVM patients and 243 Caucasian controls. SNP and haplotype association with risk of BAVM was screened using χ(2) analysis. Associated variants were further evaluated using multivariable logistic regression, adjusting for age and sex. The minor alleles of 3 GPR124 SNPs adjacent to exon 2 and localized to a 16 kb region of high linkage disequilibrium were associated with reduced risk of BAVM (rs7015566 A, P=0.001; rs7823249 T, P=0.014; rs12676965 C, P=0.007). SNP rs7015566 (intron 1) remained associated after permutation testing (additive model P=0.033). Haplotype analysis revealed a significant overall association (χ(2)=12.55, 4 df, P=0.014); 2 haplotypes (ATCC, P=0.006 and GGCT, P=0.008) were associated with risk of BAVM. We genotyped a known synonymous SNP (rs16887051) in exon 2, however genotype frequency did not differ between cases and controls. Sequencing of conserved GPR124 regions revealed a novel indel polymorphism in intron 2. Immunohistochemistry confirmed GPR124 expression in the endothelium with no qualitative difference in expression between BAVM cases and controls. SNP rs7015566 mapping to intron 1 of GPR124 was associated with BAVM susceptibility among Caucasians. Future work is focused on investigating this gene region.
RESUMEN
The orphan G protein-coupled receptor (GPCR) GPR124/tumor endothelial marker 5 is highly expressed in central nervous system (CNS) endothelium. Here, we show that complete null or endothelial-specific GPR124 deletion resulted in embryonic lethality from CNS-specific angiogenesis arrest in forebrain and neural tube. Conversely, GPR124 overexpression throughout all adult vascular beds produced CNS-specific hyperproliferative vascular malformations. In vivo, GPR124 functioned cell-autonomously in endothelium to regulate sprouting, migration, and developmental expression of the blood-brain barrier marker Glut1, whereas in vitro, GPR124 mediated Cdc42-dependent directional migration to forebrain-derived, vascular endothelial growth factor-independent cues. Our results demonstrate CNS-specific angiogenesis regulation by an endothelial receptor and illuminate functions of the poorly understood adhesion GPCR subfamily. Further, the functional tropism of GPR124 marks this receptor as a therapeutic target for CNS-related vascular pathologies.
Asunto(s)
Neovascularización Fisiológica , Tubo Neural/irrigación sanguínea , Prosencéfalo/irrigación sanguínea , Receptores Acoplados a Proteínas G/metabolismo , Animales , Vasos Sanguíneos/anomalías , Barrera Hematoencefálica/metabolismo , Movimiento Celular , Desarrollo Embrionario , Células Endoteliales/fisiología , Endotelio Vascular/embriología , Endotelio Vascular/metabolismo , Eliminación de Gen , Transportador de Glucosa de Tipo 1/metabolismo , Mesencéfalo/irrigación sanguínea , Mesencéfalo/embriología , Mesencéfalo/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Tubo Neural/embriología , Tubo Neural/metabolismo , Prosencéfalo/embriología , Prosencéfalo/metabolismo , Receptores Acoplados a Proteínas G/genética , Rombencéfalo/irrigación sanguínea , Rombencéfalo/embriología , Rombencéfalo/metabolismo , Telencéfalo/irrigación sanguínea , Telencéfalo/embriología , Telencéfalo/metabolismoRESUMEN
Vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and their receptors are important targets in cancer therapy based on angiogenesis inhibition. However, it is unclear whether inhibition of VEGF and PDGF together is more effective than inhibition of either one alone. Here, we used two contrasting tumor models to compare the effects of inhibiting VEGF or PDGF alone, by adenovirally generated soluble receptors, to the effects of inhibiting both together. In RIP-Tag2 tumors, VEGF and PDGF inhibition together reduced tumor vascularity and abundance of pericytes. However, VEGF inhibition reduced tumor vascularity without decreasing pericyte density, and PDGF inhibition reduced pericytes without reducing tumor vascularity. By contrast, in Lewis lung carcinomas (LLC), inhibition of VEGF or PDGF reduced blood vessels and pericytes to the same extent as did inhibition of both together. Similar results were obtained using tyrosine kinase inhibitors AG-013736 and imatinib. In LLC, VEGF expression was largely restricted to pericytes and PDGF was largely restricted to endothelial cells, but, in RIP-Tag2 tumors, expression of both growth factors was more widespread and significantly greater than in LLC. These findings suggest that inhibition of PDGF in LLC reduced pericytes, and then tumor vessels regressed because pericytes were the main source of VEGF. The vasculature of RIP-Tag2 tumors, in which most VEGF is from tumor cells, was more resistant to PDGF inhibition. The findings emphasize the interdependence of pericytes and endothelial cells in tumors and the importance of tumor phenotype in determining the cellular effects of VEGF and PDGF inhibitors on tumor vessels.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Factor de Crecimiento Derivado de Plaquetas/fisiología , Factor A de Crecimiento Endotelial Vascular/fisiología , Animales , Antineoplásicos/uso terapéutico , Axitinib , Benzamidas , Humanos , Mesilato de Imatinib , Imidazoles/uso terapéutico , Fragmentos Fc de Inmunoglobulinas/genética , Indazoles/uso terapéutico , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/tratamiento farmacológico , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Piperazinas/uso terapéutico , Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Pirimidinas/uso terapéutico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 1 de Factores de Crecimiento Endotelial Vascular/efectos de los fármacos , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
Intronic microRNAs have been proposed to complicate the design and interpretation of mouse knockout studies. The endothelial-expressed Egfl7/miR-126 locus contains miR-126 within Egfl7 intron 7, and angiogenesis deficits have been previously ascribed to Egfl7 gene-trap and lacZ knock-in mice. Surprisingly, selectively floxed Egfl7(Delta) and miR-126(Delta) alleles revealed that Egfl7(Delta/Delta) mice were phenotypically normal, whereas miR-126(Delta/Delta) mice bearing a 289-nt microdeletion recapitulated previously described Egfl7 embryonic and postnatal retinal vascular phenotypes. Regulation of angiogenesis by miR-126 was confirmed by endothelial-specific deletion and in the adult cornea micropocket assay. Furthermore, miR-126 deletion inhibited VEGF-dependent Akt and Erk signaling by derepression of the p85beta subunit of PI3 kinase and of Spred1, respectively. These studies demonstrate the regulation of angiogenesis by an endothelial miRNA, attribute previously described Egfl7 vascular phenotypes to miR-126, and document inadvertent miRNA dysregulation as a complication of mouse knockout strategies.
Asunto(s)
MicroARNs/genética , Neovascularización Fisiológica/genética , Proteínas/genética , Animales , Secuencia de Bases , Proteínas de Unión al Calcio , Cartilla de ADN/genética , Familia de Proteínas EGF , Regulación del Desarrollo de la Expresión Génica , Intrones , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , MicroARNs/metabolismo , Fenotipo , Proteínas/metabolismo , Vasos Retinianos/embriología , Vasos Retinianos/crecimiento & desarrollo , Vasos Retinianos/metabolismo , Eliminación de SecuenciaRESUMEN
Unlike during development, blood vessels in the adult are generally thought not to require VEGF for normal function. However, VEGF is a survival factor for many tumor vessels, and there are clues that some normal blood vessels may also depend on VEGF. In this study, we sought to identify which, if any, vascular beds in adult mice depend on VEGF for survival. Mice were treated with a small-molecule VEGF receptor (VEGFR) tyrosine kinase inhibitor or soluble VEGFRs for 1-3 wk. Blood vessels were assessed using immunohistochemistry or scanning or transmission electron microscopy. In a study of 17 normal organs after VEGF inhibition, we found significant capillary regression in pancreatic islets, thyroid, adrenal cortex, pituitary, choroid plexus, small-intestinal villi, and epididymal adipose tissue. The amount of regression was dose dependent and varied from organ to organ, with a maximum of 68% in thyroid, but was less in normal organs than in tumors in RIP-Tag2-transgenic mice or in Lewis lung carcinoma. VEGF-dependent capillaries were fenestrated, expressed high levels of both VEGFR-2 and VEGFR-3, and had normal pericyte coverage. Surviving capillaries in affected organs had fewer fenestrations and less VEGFR expression. All mice appeared healthy, but distinct physiological changes, including more efficient blood glucose handling, accompanied some regimens of VEGF inhibition. Strikingly, most capillaries in the thyroid grew back within 2 wk after cessation of treatment for 1 wk. Our findings of VEGF dependency of normal fenestrated capillaries and rapid regrowth after regression demonstrate the plasticity of the adult microvasculature.
Asunto(s)
Envejecimiento , Capilares/fisiología , Factor A de Crecimiento Endotelial Vascular/fisiología , Animales , Axitinib , Presión Sanguínea , Capilares/ultraestructura , Carcinoma Pulmonar de Lewis/irrigación sanguínea , Prueba de Tolerancia a la Glucosa , Corazón/fisiología , Imidazoles , Indazoles/farmacología , Islotes Pancreáticos/irrigación sanguínea , Riñón/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Trasplante de Neoplasias , Neoplasias Pancreáticas/irrigación sanguínea , Fenotipo , Valores de Referencia , Regeneración , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Endothelial expression of the gap junction proteins, connexin (Cx) 37, Cx40, and Cx43, varies within the vascular network. While previous studies suggest that shear stress may upregulate Cx43, it is not well understood if shear stress affects the expression of all endothelial connexins and to what extent. Endothelial cells on the upstream and downstream surfaces of cardiac valves are subjected to considerably different intensities of shear stress. We therefore reasoned that we could determine the extent hemodynamic forces affect the expression of Cx37, Cx40, and Cx43 by comparing their immunohistochemical distribution on the upstream and downstream surfaces of rat cardiac valves. We found 70- to 200-fold greater expression of Cx43 in the endothelial cells on the upstream than on the downstream surfaces. However, Cx37 was expressed almost equally in the endothelial cells on upstream and downstream surfaces, and Cx40, a major connexin in most vascular endothelial cells, was not detected on either surface. In addition to the heterogeneity in Cx43 expression, endothelial cells on the upstream surface were 35% to 65% smaller than those on the corresponding downstream surface. These results suggest that shear stress may affect endothelial cell size and Cx43 expression but not Cx37 expression.